Preparation and characterization of SrTiO3/Cu2Se p-n heterojunction
In the first stage of this work, the SrTiO3/Cu2Se p-n heterojunction was synthesized through two steps of hydrothermal process. Detailly, as illustrated in Figure 1, SrTiO3 nanoplates (NPs) (n type TE materials) were papered firstly following Cu2Se quantum dots (QDs) synthesis and in site coating on the surface of SrTiO3 NPs. After the hydrothermal process and liquid exfoliation, SrTiO3 NPs with a size of 110 nm were analyzed by transmission electron microscopy (TEM) (Figure 2a and Figure S1). Then, Cu2Se QDs, p type TE materials, were synthesized and coated on the surface of SrTiO3 NPs, forming a novel p-n heterojunction-based TE generator. The successfully coated Cu2Se QDs was obviously observed in the TEM images of SrTiO3/Cu2Se NPs (Figure 2b), in which uniform QDs decoration on the surface of SrTiO3 NPs was exhibited. The size of the prepared SrTiO3/Cu2Se NPs was appropriately increased to about 132 nm (Figure S2). Additionally, the high-resolution transmission electron microscopy (HRTEM) images with clear interference fringe and d-spacing of 0.27 nm and 0.33 nm, corresponding to the plane of SrTiO3 NPs (Figure 2c) and Cu2Se QDs (Figure 2d), provided a direct evidence for the successful fabrication of the heterojunction structure. Besides, atomic force microscope (AFM) was also applied to characterize the morphology of the fabricated SrTiO3/Cu2Se NPs. As shown in Figure 2d and 2e, a rough surface was clearly observed, which was likely attributed to the coating of Cu2Se QDs. Calculation from the 2D and 3D AFM images of SrTiO3/Cu2Se NPs, the NPs displayed a planar size of about 132 nm and thickness of 50 nm. Given that almost all thermoelectric materials have a good piezoelectric property,55-57 we determined the piezoelectric properties of SrTiO3/Cu2Se NPs by a piezoresponse force microscope (PFM), using dual alternating current resonance tracking (DART) modes with the aim to expel the displacement contribution from electrostatic interaction and topographical crosstalk in mapping the local electromechanical properties. Figure 2e-2h exhibited the topographic, vertical piezoresponse amplitude, and phase images of the SrTiO3/Cu2Se NPs, respectively. The SrTiO3/Cu2Se NPs can be clearly detected in the topographic image with clear contrast in the amplitude and phase images. Figure 2g exhibited the phase map of SrTiO3/Cu2Se NPs, which well matches the morphology map in Figure 2e. Next, a surface potential of 30 mV of SrTiO3/Cu2Se NPs was observed in the piezoelectric potential map of SrTiO3/Cu2Se NPs (Figure 2h) obtained by PFM in the darkness, which demonstrates its piezoelectric property and further testifies its thermoelectric property. To further confirm the successful fabrication and the composition of SrTiO3 NPs, Cu2Se QDs and SrTiO3/Cu2Se NPs, X-ray photoelectron spectroscopy (XPS) and X-ray diffractometry (XRD) were performed. In the XPS analysis (Figure 3a), the specific peaks of Sr, Ti, O, and Cu, Se, were exhibited in their XPS spectra, respectively. Moreover, all these characteristic peaks were observed in the XPS spectrum of SrTiO3/Cu2Se NPs. In the XRD spectrum (Figure 3b), two respective structures corresponding with SrTiO3 and Cu2Se were observed. All above observations confirmed the successful fabrication of the heterojunction structure and their potential thermoelectric property of SrTiO3/Cu2Se NPs.
The biomedical applications of nanomedicine largely depend on their physiological dispersibility and stability. It was observed that the surfaces of SrTiO3/Cu2Se NPs were slightly negatively charged after the hydrothermal process, (Figure S3), which allowed surface modification by using amphipathic DSPE-PEG through hydrophobic interaction. The zeta potential of SrTiO3/Cu2Se NPs increased to -30 mV, demonstrating a successful PEG modification and thus ensuring their physiological dispensability and stability. Around 25% (w/w) of DSPE-PEG was loaded on the surface of the SrTiO3/Cu2Se NPs as measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). PEGylation of SrTiO3/Cu2Se NPs showed improved dispersion in cell culture medium, phosphate buffer saline (PBS) and water in contrast with the bare SrTiO3/Cu2Se NPs due to lack of aggregation (Figure S4). In addition, the Fourier transform infrared (FT-IR) absorption bands of the PEGylated SrTiO3/Cu2Se NPs at ~1250 cm−1 and ~2900 cm−1 are corresponded to the C=O stretching vibration and -CH vibrations in the DSPE-PEG segment (Figures S5). Finally, Sr (red), Ti (green), and O (blue) appeared in the energy dispersive spectrometry (EDS) mapping of SrTiO3 NPs, and after Cu2Se QDs and PEG coating, Cu (purple), Se (olive), C (yellow), and N (white) showed again in the EDS mapping of PEGylated SrTiO3/Cu2Se NPs (Figure 2i), further confirmed successful surface coating by DSPE-PEG.
Next, the thermoelectric performance of our prepared SrTiO3/Cu2Se NPs were examined. The ZT of SrTiO3 NPs and Cu2Se QDs were tested and calculated, respectively. Figure 3c shows the material-dependent s as a function of temperature. It is apparently that s increases monotonically with increasing the temperature for these two samples and roughly follows a co-efficient of T-1.5, suggesting that acoustic phonons dominate the carrier scattering. Figure 3d shows the variations of S with the temperature. The positive signal of S indicates the p-type nature for Cu2Se QDs, and the negative signal of S indicates the n-type nature for SrTiO3 NPs. S increases gently upon increasing the temperature. Figure 3e presents the plots of S2s as a function of temperature, from which relatively high S2s of SrTiO3 NPs and Cu2Se QDs were obtained. Figure 3f is the plots of κ as a function of temperature, in which the relatively low κ of SrTiO3 NPs and Cu2Se QDs were also obtained. Due to the obtained high S2s as well as low κ, significantly enhanced ZT values are expected. Figure 3f is the ZT plots as a function of temperature, in which a peak ZT of 0.11 and 0.17 at 333 K is achieved in the fabricated SrTiO3 NPs and Cu2Se QDs. Figure 3h shows the S at room temperature as a function of the natural logarithm of s of SrTiO3 NPs and Cu2Se QDs. This linear relationship between the S and the natural logarithm s indicates more fluctuating carrier concentration and less varying carrier mobility. All above observations confirmed the good thermoelectric properties of SrTiO3/Cu2Se NPs.
SrTiO3/Cu2Se NPs mediated reactive oxygen species (ROS) generation
Followingly, the light-heat-electricity-chemical energy conversion of SrTiO3 NPs, Cu2Se QDs, and SrTiO3/Cu2Se NPs were measured and analyzed. Figure 4a and 4b show the light-heat conversion of SrTiO3/Cu2Se NPs, in which the temperature increased from 35 oC to 45 oC under 2.5 min 808 nm laser irradiation and cooled down to 35 oC following 9 min natural cooling process. As shown in Figure 4h, according to thermoelectric effect, under temperature gradient (35 oC - 45 oC) induced by 808 nm laser irradiation and natural cooling, a build-in electric field was constructed on the opposite surfaces of SrTiO3 NPs or Cu2Se QDs. Therefore, the built-in electric field can facilitate separation of charges (electrons and holes) in bulk, and promote their transfer to the catalyst surface, making them effective tools for catalyzing reduction and oxidization of O2 and H2O to generate superoxide anion (·O2−) and hydroxyl radical (·OH), respectively. ·O2− generation through the reduction of electrons was measured with dihydrorhodamine 123 (DHR 123) probe. As shown in Figures 4c and 4e, obvious fluorescence increase was detected following the temperature gradient (35 oC - 45 oC) using SrTiO3 NPs and Cu2Se QDs separately, which indicated that SrTiO3 NPs and Cu2Se QDs are suitable nanomedicines for thermoelectric therapy. Of note, a much stronger fluorescence increase was observed when SrTiO3/Cu2Se NPs were applied as thermoelectric catalysts for catalyzing ·O2− generation from O2. As exhibited in Figure 4h, after SrTiO3 NPs (n type) contacting with Cu2Se QDs (p type), an interfacial electric field was then constructed on their interface, in which the separated electrons and holes induced by thermoelectric effect further transferred and re-located on the surface of different catalysts following interfacial electric field. Thereby, the recombination of electrons and holes was restricted, leading to extending lifetime of separated electrons and holes for further redox reactions. The p-n heterojunction enhanced thermoelectric effect of SrTiO3/Cu2Se NPs was further assessed in terms of hydroxyl radical (·OH) production using methylene blue (MB) as the specific probe of ·OH. Consistent with the above results, the SrTiO3/Cu2Se NPs exhibited the strongest ·OH generation, which further confirmed their p-n heterojunction enhanced thermoelectric effects (Figure 4d and 4f). By employing 5,5-dimethyl-1-pyrroline N-oxide as the spin trapping agent, electron spin resonance (ESR) was applyed to detect the gnerated ROS directly. As shown in Figure 4g, ·O2− and ·OH synchronously generated from SrTiO3/Cu2Se NPs through thermoelectric effects from O2 and H2O were detected, which further confirms the high ability of ROS generation of SrTiO3/Cu2Se NPs.
In vitro antitumor evaluation mediated by SrTiO3/Cu2Se NPs
The biocompatibility of the prepared TE agents was next tested using MCF 7 and Hela cancerous cells. As shown in Figure 5a and S7, similar with the traditional photothermal agent (graphene, G), the TE agents showed negligible cytotoxicity in the absence of excitation, and more than 80% of the cells were viable even when exposed to 100 mg/mL of the respective TE agents. Stimulation with 808 nm laser led to the temperature increasing from 35 oC to 45 oC, and thus increased the cytotoxic effects of all TE agents (Figure 5b and S8). However, under the uniform 808 nm laser irradiation and temperature increase, the cells treated with G still remain a relatively high viability.To this end, we speculated that the cytotoxic effects of TE agents are probably attributed to the thermoelectric effect inducing ROS generation, rather than photothermal effect inducing heat. With increasing cycles of this temperature gradient (35 oC - 45 oC), an enhanced cell cytotoxic effects of TE agents were observed, meanwhile, the cytotoxic effects of G remain negligiblely improved. This observation further demonstrated the thermoelectric effect of TE agents inducing ROS generation was the main cause for their cytotoxicity. Moreover, the cells treated with SrTiO3/Cu2Se NPs exhibited the highest cytotoxicity, confirming the p-n heterojunction enhanced thermoelectric effect. Additionaly, the intracellular ROS levels under different treatments were analyzed by using fluorescent probe. Indeedly, the intracellular ROS levels were signficantly higher in the SrTiO3 NPs and Cu2Se QDs treated groups, compared to those treated with G, and the highest ROS concentration was detected in cells exposed to the SrTiO3/Cu2Se NPs coupled with 808 nm laser irradiation and a natural cooling process (Figures 5d and 5e). As previously reported,58 DNA damage caused by ROS is one of the main causes for ROS-induced cell toxicity. Thus, the levels of DNA damage in MCF 7 cells after different treatments were further analyzed using γ-H2AX as a marker for DNA double-strand breaks. As shown in Figure 5f, MCF 7 cells treated with G with DT (35 oC - 45 oC) did not show any obvious DNA damage compared with the control group. However, MCF 7 cells treated with SrTiO3 NPs or Cu2Se QDs coupling with the same DT, showed apparently detectable levels of DNA damage, further supporting the TE effect of SrTiO3 NPs and Cu2Se QDs. Treatment with SrTiO3/Cu2Se NPs and DT produced considerably high levels of irreparable DNA damage in the cancer cells. All these results thus suggest that the developed therapeutic strategy based on SrTiO3/Cu2Se NPs and DT with an enhanced TE effect can specifically kill cancer cells. Moreover, the efficient apoptosis of SrTiO3/Cu2Se NPs via TE effect was further detected through co-staining cells with propidium iodide (PI, dead cells, red fluorescence) and calcein AM (live cells, green fluorescence) after different treatments. The LSCM images of co-stained cancer cells, as shown in Figure 5g, also confirmed the antitumor effect of TET in vitro.
In vivo antitumor evaluation mediated by SrTiO3/Cu2Se NPs
The anti-cancer potential of TET was next evaluated in vivo using MCF 7 tumor-bearing mice. The mice were each injected intravenously with Cy7-labeled NPs at the dosage of 5 mg/kg, and the fluorescence intensity of Cy7 in the blood was measured at different time intervals. As shown in Figure 6b, the Cy7-loaded NPs remained signficantly longer in circulation compared to free Cy7, which was suggestive of greater tumor accumulation of NPs. Additionally, the tumor accumulation of NPs was also confirmed by fluorescence imaging of major organs after 24 h i.v. injection (Figure 6c). To more precisely characterize the biodistribution of NPs in vivo, an ICP was employed to test the concentration of NPs in the major organs and tumors over 24 hours, which also showed a great tumor accumulation of the prepared NPs. The MCF 7 tumor-bearing mice were randomly divided into the following groups and treated accordingly: 1) saline control, 2) SrTiO3/Cu2Se NPs, 3) SrTiO3 NPs + DT, 4) Cu2Se QDs + DT, 5) SrTiO3/Cu2Se NPs + DT, 6) G + DT, and 7) PTT (G, >55oC). The DT means temperature gradient (35 oC - 45 oC) for 3 cycles inducing by 808 nm laser irradiation and natural cooling after 24 h post injection (Figure 6a and 6e). The tumor volume was measured every 2 days, and as shown in the growth curves in Figure 6f and 6g, the untreated and non-DT controls did not show any significant inhibition of tumor growth. The combination of TE agents (SrTiO3 NPs or Cu2Se QDs) and DT achieved an obvious inhibitory effect, which is attributed to the generation of ROS by the thermoelectric effect. Due to the p-n heterojunction enhanced thermoelectric effect, SrTiO3/Cu2Se NPs with DT markedly inhibited tumor growth, in which SrTiO3/Cu2Se NPs almost completely ablated the tumors under the same conditions. In contrast, the G NSs with the same DT treatment exhibited only a slight inhibition of tumor growth compared with the control group, which indicated the temeperature of 45oC can not able to induce cancer cells death. Only the hyperthermia (>55 oC) induced by G NSs-mediated PTT could provide a similar anti-tumor effect as that of TET (45 oC) (Figure 6h). However, as shown in Figure 6g, because the hyperthermia (>55 oC) randomly propagated and diffused to the surrounding tissues, an obvious PTT-related toxicity and side effects on normal tissues was observed, in which the skin and muscle at the 808 nm laser irradiation site were scorched. By contrast, there was negligible damage to the skin at the irradiated sites of TET (45 oC) (Figure 6f). These findings are consistent with the thermoelectric effect and heterojunction structure of SrTiO3/Cu2Se NPs that induces an intracellular ROS burst. The representative images of mice from the different treatments are shown in Figure 6f and 6g. Moreover, the mice treated with SrTiO3/Cu2Se NPs + DT showed the longest lifetime without any tumor recurrence (Figure 6l). No signficant changes were observed in the body weight of the mice during the experimental period (Figure S10), indicating negligible adverse effects of this therapy in vivo.
The intracellular ROS burst effect was further validated by using DCFH fluorescence probe. As shown in Figure 6i and S11, the different treatments led to consistent ROS accumulation in the tumors, and the strongest green fluorescence was detected in the SrTiO3/Cu2Se NPs + DT group, further indicating a drastic ROS burst in tumor cells. Given that ROS induce apoptosis through DNA damage,58 we next analyzed the tumors for signs of DNA double strand breaks, oxidative stress and apoptosis. SrTiO3/Cu2Se NPs alone induced negligible γ-H2AX foci and very few apoptotic cells, whereas both DNA damage and apoptosis were considerably higher under SrTiO3 NPs or Cu2Se QDs with DT. Coupling SrTiO3/Cu2Se NPs and DT led to a marked increase in γ-H2AX foci and apoptosis in the tumors (Figure 6j and 6k). Furthermore, the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidized DNA, were consistent with that of γ-H2AX (Figure S12). Taken together, the p-n TE agents heterojunction can efficiently trigger a ROS burst in cancer cells and induce apoptosis under a mild condition.
Biocompatibility evaluation of SrTiO3/Cu2Se NPs
The biocompatibility of TET was evaluated via hematological, histological and immunological indices. As shown in Figure 7a-7c, no obvious difference in ROS levels, DNA damage, apoptosis, and tissue damage were observed in normal tissues after treatment with SrTiO3/Cu2Se NPs + DT compared with those treated with PBS, indicating a favorable biocompatibility of the SrTiO3/Cu2Se NPs and TET in normal tissues. Moreover, the serum levels of IFN-γ, IL-6, TNF-α and IL-12+P40 were similar in the control and treated mice 12 and 24h post i.v. injection of SrTiO3/Cu2Se NPs (10 mg/kg) (Figure 7d). In addition, routine blood examination on days 1, 7 and 14 post-injection did not show any significant differences in aspartate aminotransferase (AST), alanine aminotransferase (ALT), white blood cells (WBC), blood urea nitrogen (BUN), alkaline phosphatase (ALP), red blood cells (RBC), platelet (PLT), Hemoglobin (HGB), mean corpuscular volume (MCV), creatinine (Cr), lymphocyte (LYM), hematocrit (HCT), and neutrophil (NEU) between the control and treated groups (Figure 7e). Taken together, SrTiO3/Cu2Se NPs and TET are biocompatible in vivo.
Comparation of side effects between PTT and TET
As demonstrated above, PTT with hyperthermia (>55 oC) could easily damage the skin at irradiated site. To further confirm the superiority of TET, we finally compared the treatment-related toxicity and side effects on normal tissues and organs through simulating the PTT and TET at some major organs and tissues. As exhibited in Figure 8, exposure to the TET (45 oC) conditions, negligible toxicity or side effect are observed in their H&E staining images of heart, liver, spleen, lung, kidney, muscle, and skin, compared with these without any treatment. However, obvious and serious damages were revealed in these important organs and tissues after exposing to the PTT (55 oC) conditions. For example, congestion, enlargement of intercellular space, tissue defect, etc, were presented in these major organs. Additionally, evident swelling and critical damage were also observed in muscle and skin under treated with PTT (55 oC). All above phenomena further confirmed the in vivo safety of TET and demonstrated competitive advantages over PTT.